Topic

Traceability of genetically-modified plants (GM plants) will play an important part in quality control and monitoring of primary and processed plant products in future – as the number of GM plants increases, the same genetically-introduced DNA constructs are likely to occur in different cultivars. High cross-fertilisation rates in rape, an agricultural crop plant, make the transfer of transgenic DNA sequences to conventional cultivars probable.

In addition to the detection of transgenic DNA sequences, differentiation of the rape cultivar spectrum is becoming increasingly important in identifying the cause of undesirable GMO contamination in the harvest and in processed products. This project is developing molecular biological procedures to provide fast and inexpensive genotyping of GM rape lines. The data are collected in a fingerprint database to be made available to Bavarian monitoring laboratories.

Experiment description

Identification of GM rape cultivars and their cross-bred progeny is carried out using DNA-based detection methods. Compared to conventional methods, DNA methods have the advantage of making it possible to trace the GM cultivar at any stage in a plant’s life and also in processed plant products. It is possible to analyse extremely small amounts of mixed samples such as are found in the food and feed industry. Identification of GM rape cultivars is based on evidence of the introduced DNA construct that gives the plant its transgenic characteristics, and also on identification of the rape cultivar using molecular DNA markers. Miniaturised DNA microchips that cover the entire spectrum of German rape cultivars are being developed to ensure a high sampling rate at low cost.

Detection of the transgenic DNA construct

The DNA sequences of the DNA construct currently used for transformation of rape cultivars are available from public databases. Based on these sequences, PCR methods (polymerase chain reaction) are to be developed for DNA detection. The PCR procedure is based on reproduction of the desired DNA sequence using the DNA polymerase enzyme. During the enzymatic PCR reaction, only the target DNA is reproduced exponentially, making it possible for even extremely small GMO levels (<0.1%) to be detected in mixed samples.

Microsatellites are DNA fragments in which a short DNA sequence is repeated several times. The number of repetitions can vary between individuals of a species. The specific microsatellite pattern is then inherited by the progeny. For this reason, microsatellites have for some time been used as DNA markers in paternity tests. Microsatellites are favoured for genotyping in plant genetics because of the large amount of information they provide. Numerous microsatellite markers for rape are now available from public databases. This project aims to characterise the current spectrum of German rape cultivars using the most informative microsatellite markers.

Identification of rape cultivars by means of DNA point mutation

A mutation leading to a single nucleotide exchange in the DNA sequence of an organism is called a point mutation, or single nucleotide polymorphism (SNP). As there are four nucleotides in total, there are four possible expressions of an SNP. Thus SNP markers possess a high degree of polymorphism compared with other DNA markers. SNP markers have been used for some time in human genetics because of the frequency with which they occur in the genome. This project aims to identify SNP markers in the rape genome by taking DNA sequences for rape genes from internet databases and using sequence analysis to test for the presence of SNP. It is difficult to identify SNP within the complex rape genome – made up of the genomes of turnip rape and cabbage – since similar sequences occur several times in the genome as a result of the combination of the two contributing genomes.

Creation of a DNA microchip to trace GM rape cultivars

As well as identifying rape cultivars, the DNA microchip will also provide evidence of transgenic sequences. High sampling rates, low use of materials and low labour intensity are made possible by this miniaturising process. Short, single-stranded DNA sequences (oligonucleotides) are fixed on the surface of the DNA chip. These sequences extract the target DNA from the sample, thus triggering enzymatic reproduction of the target DNA through a polymer chain reaction. The nucleotides used as building blocks in DNA reproduction are marked with various fluorescent dyes. A four-colour chip reader can detect the fluorescent marking of the nucleotide integrated at the SNP site.

Setting up a database

After collecting the marker data, a fingerprint database is to be set up that will also include construct-specific characteristics of GM rape cultivars. Detailed information on the composition of the transgenic constructs (regulatory sequences, structure and selection genes) allows quick identification of GM rape cultivars and accurate traceability of adventitious cross-pollination and varietal mixtures. The database will be made available to the Bavarian monitoring laboratories, and will provide accurate and inexpensive traceability of transgenic rape and its processed products.

Results

Detection of the transgenic DNA construct

A DNA microchip that can detect transgenic sequences has been developed. As well as aiding detection of specific plant species (maize, soya or rape), this bio-chip can also aid detection of various frequently-used transgenes such as pat, bar, epsps, nptII etc.

Identification of rape cultivars by means of microsatellites

16 microsatellites considered suitable for the identification of rape cultivars were chosen from the scientific literature. Nine of these microsatellites were sufficient to allow one hundred percent differentiation of rape cultivars. Seven more microsatellites were analysed for good measure.

Identification of rape cultivars by means of DNA point mutation

So far, cultivar-specific point mutations have been examined in 140 rape DNA fragments (around 73 kb). 51 of these sequences proved to be genome specific. The remaining sequences were not tracked any further. The genome-specific DNA fragments were then examined for evidence of point mutations. There was evidence of point mutations in 16 fragments. The type, position and number of point mutations in these DNA fragments were accurately determined by means of sequence analysis. To date a total of 19 SNP markers suitable for differentiation of rape cultivars have been detected from these DNA fragments. These 19 SNP markers make it possible to identify 61 different conventional German rape cultivars.

Traceability of GM rape cultivars

Traceability of four transgenic rape lines is also possible with these 19 SNP markers.

Creation of a DNA microchip to trace GM rape varieties

Building on the data collected from sequence analysis of the SNP markers, a DNA microchip is now to be created to make traceability of GM rape cultivars possible.